摘要
目的探讨降钙素基因相关肽（CGRP）对高氧暴露下肺泡Ⅱ型上皮细胞（AECⅡ）的促增殖作用及其蛋白激酶Cα／核转录因子-κB（PKca／NF—κB）信号途径的调控机制。方法将原代分离培养的孕21d胎鼠AECⅡ分为空气组、高氧组、高氧CGRP组、高氧CGRP拮抗剂组。空气组和高氧组分别在21％0z或85％。中暴露24h；高氧CGRP组在高氧处理前加入CGRP，高氧CGRP拮抗剂组同时加入CGRP和CGRP受体拮抗剂CGRP8—37。采用四甲基偶氮唑盐（MTT）比色法和流式细胞术测定细胞增殖能力和不同细胞周期细胞比例；蛋白质免疫印迹法（Westernblotting）检测胞膜和胞质PKCa的表达；激光共聚焦检测NF—κBp65的细胞核表达。结果高氧组G0／G1期细胞比例明显高于空气组[（80．652±6．253）％比（45．825±2．899）％]，而细胞增殖率[（68．752±5．766）％比（100．000±6．682）％]及S期、G2／M期细胞比例[分别为（14．198±4．785）％比（27．470土2．775）％，（5．148±1．688）％比（26．708±1．863）％]均低于空气组（均P〈0．01）。CGRP干预可提高高氧暴露AECⅡ的增殖能力[（94．813±6．102）％]，使s期和G2／M期细胞增多[（30．547±9．861）％，（17．668±9．509）％，均P〈0．013。高氧组胞膜与胞质PKCa比值显著低于空气组（0．63±0．10比1．00±0．09），而NF—KBp65的荧光强度高于空气组（22．98±2．20比14．54±2．35）；高氧CGRP组胞膜与胞质PKCa比值（1．41±0．23）及核内NF—KBp65荧光强度（35．38±3．37）均高于高氧组（0．63±0．10，22．98±2．20）及高氧CGRP拮抗剂组（O．74±0．10，24．88±1．81，均P〈0．01）。结论cGRP可促进高氧暴露下AECⅡ的生长增殖；PKCα参与了CGRP对细胞作用的信号传递，而NF—κB是PKCα的下游分子之一，仅部分执行PKCα的效应。
Objective To explore the effects of calcitonin gene-related peptide （CGRP） on type alveolar epithelial cell （AEC I ） exposed to hyperoxia, and to determine whether the mechanism is mediated by protein kinase Ca/nuclear factor-κB （PKCα/NF-κB） signal pathway. Methods AEC Ⅱ were isolated from the lung of 21 days fetal rat and cultured for 15 hours to coalesce. Then AEC Ⅱ were randomly assigned into four groups., air, hyperoxia, O2/CGRP, and O2/CGRP8-37 （a receptor antagonist against CGRP）. AEC Ⅱ were exposed to FiO2 21% （air） or 85% （hyperoxia） for 24 hours respectively. In OJCGRP and O2/CGRPS-37 groups CGRP or both CGRP and CGRP8-37 were added into cultural fluid before placing the plate into 85% oxygen. Cell proliferation ability was determined by methl thiazolyl tetrazolium （MTT） assay and cell cycles by flow cytometry. Western blotting was employed to detect the fraction of PKCa in membrane and cytosol, and translocation of NF-κB was observed under laser confocal microscopy. Results AEC Ⅱ in hyperoxia group showed a decreased viability of AEC Ⅱ （（68. 752 ± 5. 766）% vs. （100. 000± 6. 682）%3 and had an enhanced percentage of G0/G1 phase （（80. 652±6. 253）% vs. （45. 825±2. 899）%3 with a corresponding decline in percentage of S phase （（14. 198 4-4. 785）% vs. （27. 470±2. 775）%3 and G2/M phases （（5. 148±1. 688）% vs. （26. 708±1. 863）% compared with AEC Ⅱ in air （all P〈0.01）. Addition with CGRP before hyperoxia exposure promoted AEC I proliferation （94. 813±6. 102）% and enhanced the cell proportions in S and G2/M phases （（30. 547± 9. 861）% and （17. 668±9. 509）%, all P〈 0. 01）. The ratio of membrane to cytoplasm fraction of PKCa declined （0. 63±0. 10 vs. 1.00±0. 09） and the fluorescence of NF-κB in nucleus enhanced （22.98±2.20 vs. 14.54±2.35） in hyperoxia compared with that in air, while both the ratio of PKCα and intensity of NF-κB were increased in O2/CGRP group （1.41-t-0. 23, 35.38＋ 3.37） compared with those in hyperoxia （0.63 -t- 0. 10, 22.98 ：J： 2.20） and O2/CGRP8-37 groups （0. 744±0.10, 24.88±1.81, all P〈0. 01）. Conclusion CGRP could promote proliferation of AEC Ⅱ when exposed to high oxygen tension. PKCα participates in the signal transduction process and NF-κB is a downstream molecular of PKCα, executing in part the function of PKCα signal.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2010年第5期263-266,I0001,共5页
Chinese Critical Care Medicine
基金
基金项目：国家自然科学基金面上项目（30670931）
云南省应用基础研究项目（2008ZC083M）
